Background information concerning various aspects of this invention may be found in the patents and publications incorporated in this disclosure by reference and listed at the end of the specification. The parenthetical numerals appearing in the following discussion refer to the respective patents and publications.
The process of blood coagulation is an important activity which normal, whole blood is capable of carrying out under timely circumstances to prevent excessive loss of blood through open wounds or by internal bleeding. It is known that normal whole blood contains a factor which is absent, or seriously deficient in hemophiliacs. This factor is associated with the globulin fraction of blood and has come to be known as antihemophilic factor, AHF, FACTOR VIII, or AHG.
Scientists have known about AHF and its role in blood coagulation for some time, and the treatment of hemophilia heretofore has generally consisted of replacement therapy whereby the patient is transfused with many pints of fresh whole blood or specially prepared plasma.
It is known, however, that under ordinary storage conditions whole blood and liquid plasma begin rapidly to lose their AHF activity in a day or so. While it is possible to freeze and store fresh plasma, AHF activity in frozen plasma may also decrease with time. It is possible to reduce this loss of AHF activity by storing or drying freshly frozen plasma, but it is desirable to have a source of AHF of known strength and increased potency.
Many methods of isolation or concentration of AHF for the preparation of plasma fractions rich in AHF from human or animal blood have been made, and certain methods have been developed which are reliable, in that AHF activity of a desired concentration has been produced.
In preparation of cryoprecipitate and the concentration of AHF in this material is known (16). Cryoprecipitate concentrate refers to the precipitate obtained from the freezing and cold thawing of human or animal blood plasma, and separated from the supernatant fraction of the plasma. The cryoprecipitate concentrate is preferably obtained by the rapid freezing of fresh plasma, although stored plasma can also be used. The freezing is usually carried out at temperatures from about -20.degree. C. to about -40.degree. C., followed by slow thawing at about 4.degree. C. Many precipitating agents for AHF are known. These include ethanol, ethyl ether, ammonium sulphate, phosphate-sodium citrate, amino acids, glycine, and others (16).
One of the important precipitating agents for AHF is a substance known as polyethylene glycol, abbreviated as PEG. Polyethylene glycol is obtained by the polymerization of ethylene oxide in the presence of ethylene glycol (Ref. 1; Vol. 10, pp. 638-676). Polyethylene glycols, also known as poly(oxyethylene) are prepared in various molecular weights ranging from the liquid polyethylene glycols having a molecular weight of from about 200 to about 600 and the solid polyethylene glycols having molecular weights from about 900 to about 8,000, with paste-like materials in between about 600 and 900. Polyethylene glycols are sold by Union Carbide under the name CARBOWAX and UCON, the latter being a liquid product.
Closely related to polyethylene glycol chemically and in properties are the polypropylene glycols and the mixed polyethylene-polypropylene glycols, the latter being block copolymers of ethylene oxide and propylene oxide, a large group of which are sold under the trade name PLURONIC, by BASF Wyandotte Chemical Company. (1; Vol. 10, pp. 658-659 and Ref. 2). These classes of compounds are known for their detergency and for their lack of toxicity (1,2) and both have been studied quite extensively in biological systems.
Polson studied the use of polyethylene glycol for the purification of proteins (9) and fractionation of blood plasma (10, 11, 12). Extensive studies on the use of PEG intravenously have been conducted (13, 19, see also 2-8). The effect of PEG on AHF concentrates has been studied quite extensively and there have been a number of improved blood fractionation procedures (14,15) including a procedure for preparing a stable high potency human AHF concentrate using PEG and glycine to fractionate a cryoprecipitate of AHF concentrate (16). PLURONIC polyols, because of their close chemical structure, their known lack of toxicity (1), and their proven compatibility with cells (3-6) make this class of compounds an obvious substitute for PEG in plasma fractionation procedures where PEG has been successfully used.
The chemistry and the anticoagulant effects of heparin are well known and heparin has been used in fractionation procedures (17). Heparin is said to inhibit the conversion of prothrombin to thrombin (18). The addition to plasma fraction to prevent coagulation, on a micro as well as a macro scale, is an obvious expedient. It has been found, however, that heparin alone is not entirely effective in preserving protein fractions, such as blood coagulation factors, when plasma or plasma fractions are processed at or near normal room temperature.
A number of advantageous features respecting this invention flow from the discovery that if heparin and a polyol, such as polyethylene glycol or a PLURONIC polyol or combination of these, are combined with plasma before freezing to produce cryoprecipitate. Blood factors such as AHF are preserved and greatly increased yields can be obtained and a product of greater stability and higher potency can be obtained than has been available in the prior art. An apparently synergistic preservative effect exists between heparin and a polyol such that plasma, plasma fractions and blood factors are preserved more effectively than by the additive effect of individual constituents alone. By appropriate utilization of this heparin-polyol combination, significant increases in the yield of AHF during cryoprecipitation and subsequent fractionation can be obtained.
AHF recovery depends to a significant extent upon the temperatures utilized in the concentration process. While it is obviously convenient to operate at or near room temperature, prior art room temperature process steps had to be carried out very quickly because AHF degrades very rapidly at room temperature and always involves significant loss of AHF. The preservative effect of heparin and polyol (PEG or PLURONIC polyol) is particularly important in processes in which the plasma fraction, including whole plasma, is processed at or near room temperature or when processing of the plasma fraction is prolonged. Unlike the prior art in which losses increase with increasing processing time, processing time becomes of secondary importance once the preservative effect of the present invention has been achieved.
The present invention is an improvement in the process of collecting and concentrating blood coagulation factors, wherein blood plasma is frozen and then thawed for further concentration or use.
The invention comprises adding heparin and a material selected from the group consisting of polyethylene glycol and alpha-hydroxy-omega-hydroxy-poly(oxyethylene)poly(oxypropylene) poly(oxyethylene) block copolymer or mixtures of the same to the blood plasma.
One feature of this invention is that AHF and other blood factors are preserved by adding a preservative solution to plasma containing such blood factors. Therapeutic agents for replacement therapy in hemophilia can thus be manufactured in higher yields when the preservative solution is used.
Another feature of the invention is that there is provided a preservative solution which consists essentially of heparin and either polyethylene glycol or alpha-hydroxy-omega-hydroxy-poly(oxyethylene)poly(oxypropylene)poly(oxyeth ylene) copolymer, or both, in a suitable buffer solution, such as a citrated saline solution.
A related feature of the invention is that the preservative solution can be used to great advantage by mixing the preservative solution with plasma soon after collection to preserve the AHF and/or other protein constituents of the plasma. The plasma is frozen and, when cold thawed, the cryoprecipitate therefrom may be used as a source of AHF and other constituents.
In one of its more important usages, the invention may be described similarly as a process of concentrating blood coagulation factors from blood plasma which includes the steps of freezing the blood plasma to obtain a cryoprecipitate fraction, separating the cryoprecipitate fraction from the bulk of the plasma, and concentrating the blood coagulation factors in the fraction by precipitation and dissolution steps which is improved by adding a preservative solution to the blood plasma before freezing the plasma. The preservative solution consists essentially of heparin and a polyol selected from a group consisting of polyethylene glycol and a PLURONIC polyol of the class alpha-hydroxy-omega-hydroxy-poly(oxyethylene) poly(oxypropylene)poly(oxyethylene) block copolymers, or mixtures of these polyols. Following thawing, the cryoprecipitate may be handled in any conventional manner and the AHF may be concentrated in any conventional manner, such as by fractionation with alcohol, glycine and PEG, etc.
In the preferred embodiment, the method comprises adding the preservative solution to plasma immediately upon collection, then causing the formation of cryoprecipitate by freezing and cold thawing the preserved plasma. That cryoprecipitate may be used directly or further processed to produce AHF concentrates.